1. Field of the Invention
The present invention relates to inhibitors and probes of protein phosphatases. In particular, the present invention relates to unnatural amino acids that mimic phosphotyrosine.
2. Description of the Prior Art
It is generally known that probes can be used to target or identify various biological compounds to determine the presence of a disease state or provide therapy. Probes bound with a biological compound of interest can be detected by various methods such as fluorescent imaging or assays.
U.S. Patent Application Publication No. 2007/0009428 to Syud, et al. discloses diagnostic compounds designed for use in a pretargeting strategy comprising a ligand and an enzyme. A set of compounds comprising an active agent-labeled species and a pretargeting conjugate is disclosed. The active agent-labeled species includes a ligand coupled with an active agent selected from a group consisting of diagnostic active agents, therapeutic active agents, and combinations thereof. The pretargeting conjugate includes a protein that is conjugated to a targeting species having a targeting moiety capable of binding to an in vivo target or a biomarker substance produced by or associated with the target. The protein is substantially free of a cofactor.
International Patent Application Publication WO/2005/050226 to Peters, et al. discloses fluorous-based methods and compositions for preparation, separation and analysis of complex biologically-derived samples, such as proteomic and metabolomic samples. A fluorous labeling reagent is provided comprising a chemically-reactive functional group coupled to a fluorous moiety comprising five or more fluorine atoms. The fluorous labeling reagent is coupled to one or more member compounds in the biologically-derived sample, via the chemically-reactive functional group, to produce fluorous labeled sample members, thereby preparing the biologically-derived sample for analysis. The biologically-derived sample can be, for example, a proteomics sample or a metabolomics sample; exemplary sample sources include, but are not limited to, cell lysates, cell secretions, tissue samples, bodily fluids such as blood, urine, or saliva, and the like. In addition to targeting naturally-occurring chemical moieties in a select sample, a reactive functionality can be introduced into the biologically-derived sample to facilitate the fluorous labeling.
Protein phosphatases are regulatory enzymes implicated in signal transduction pathways and diseases such as diabetes, obesity, and cancers. Phosphatases act in opposition to protein kinases and remove phosphate groups added on by the kinases to restore proteins to their dephosphorylated state. There are several different types of protein phosphatases classified according to the substrate that they act on. For example, serine/threonine specific phosphatases remove phosphate groups from phosphorylated serine and threonine. Tyrosine specific phosphatases (protein tyrosine phosphatases—PTPs) remove phosphate groups from phosphorylated tyrosine, a central regulatory mechanism for cellular signal transduction. PTPs can also have dual specificity. There are also low molecular weight PTPs. PTPs have been implicated in cellular growth and differentiation, mitotic cycles, metabolism, motility, cytoskeletal organization, neuronal development, cell-cell interactions, gene transcription, immune response, and oncogenic transformation. PTPs catalyze dephosphorylation reactions by forming a phospho-enzyme intermediate.
PTP1B functions as a positive regulator of signaling events associated with breast and ovarian tumorigenesis, in addition to playing a role in down-regulating insulin and leptin signaling. Another PTP, PTP4A3, is implicated in cell proliferation, migration, and cancer metastasis. Since there are more than 100 PTPs in humans, it is desirable to clearly define the partnerships between individual PTPs and phosphoproteins.
U.S. Patent Application Publication No. 2005/0233469 to Zhang, et al. discloses compounds capable of covalently binding to a protein tyrosine phosphatase (PTP) for use in tracking PTP activity as well as identifying and isolating PTPs. The invention is also directed to methods of identifying a PTP involved in a disease in a mammal. The methods comprise obtaining a first cellular extract from a mammal that has the disease and obtaining a second cellular extract from a mammal that does not have the disease; combining each cellular extract with one of the above compounds, where R is a reporter moiety; and assessing (e.g., quantifying) PTPs in each cellular extract by assessing (e.g., quantifying) the amount of reporter moiety bound to each PTP. The presence of a greater amount of a PTP in one of the cellular extracts over the other cellular extract indicates that the PTP is involved in the disease. The present invention utilizes natural amino acid-like structures, which can maximally harness the specificity of protein (tyrosine) phosphatases towards their naturally partnering protein substrates, which contain phosphoamino acids. The compounds disclosed in the present invention, in addition to the applications of the compounds in the previous invention, can be incorporated into proteins, peptides or analogs to identify protein phosphatases that dephosphorylate specific phosphopeptides or phosphoproteins by replacing the corresponding phosphoamino acids.
Activity-based probes have been used to label enzymes including phosphatases. Among activity-based PTP-targeting probes, shown in FIG. 16, turnover-based suicidal substrates containing fluoromethylphenyl phosphate (e.g., 1 and 2) have been intensively studied (Myers, J. K., 1993, among many others). Hydrolysis of such substrates by PTPs or other phosphatases generates a highly reactive quinone methide, which then reacts with a nucleophile near the phosphatase active site. More recently, the derivatives of α-bromobenzylphosphonate (3) and phenyl vinylsulfonate (4) have been used as class-specific PTP probes (Kumar, et al. 2004). However, these probes are reactive towards thiols even in the absence of PTPs, especially in neutral or basic aqueous solutions, and thiols are commonly present in the reducing cellular environments. Furthermore, α-bromobenzylphosphonate undergoes solvolysis under similar conditions.
While substrate-trapping mutants of PTPs can be used to identify their physiologically relevant substrates, no methods are available to allow direct identification of PTPs for phosphopeptide substrates. Currently, no methods are available for identifying protein phosphatases that dephosphorylate specific phosphopeptides or phosphoproteins. Therefore, there is a need for probes that can detect such protein phosphatases.